Chymosin (also called rennin E.C. 3.2.23.4) is an enzyme isolated from the fourth stomach of the calf. It is valued for its ability to produce milk curds by catalyzing the hydrolysis of kappa-casein to para-kappa-casein which, in the presence of calcium ion, precipitates as the insoluble curd calcium paracaseinate. After removal of the whey, the curd is processed to make various types of cheeses. Although several proteolytic enzymes will clot milk, the best cheeses have been produced from milk clotted with preparations rich in chymosin. The availability of chymosin--and ultimately its price--depends upon the demand for veal, since the production of chymosin diminishes, and the production of pepsin increases, as the animal matures and is weaned. Enzymes similar to chymosin have been extracted from microorganisms fungi, especially Mucor mehei, but the curd and, ultimately, the taste of the cheese are different. The superiority of chymosin for cheese making is probably related to the highly specific manner in which it attacks its substrate, kappa-casein.
Bovine calf chymosin exists as two isozymes (designated A and B), which can be resolved by DEAE-cellulose chromatography of the crystalline enzyme. Isozyme B, which may be catalytically less efficient than isozyme A, is the more abundant form in tissue extracts. Bovine calf chymosin isozyme B has been sequenced, and isozyme A has been partially sequenced, to reveal only one amino acid difference at residue 290 (glycine in B and aspartic acid in A) (see Foltmann, B. et al., J. Biol. Chem., 254: 844-8457 [1976]).
Chymosin is synthesized within the cells of the stomach lining in a precursor form known as preprochymosin. The "pre" portion of preprochymosin is a sequence of amino acids located at the amino terminus. These amino acids comprise a signal peptide which appears to be involved in the transport of the protein to the cell wall for secretion into the periplasmic space. The signal peptide is cleaved at the cell wall and the enzyme is secreted as prochymosin. Prochymosin is a zymogen containing 365 amino acids (40,477 Daltons). Prochymosin is converted to chymosin by the specific removal of 42N-terminal amino acids. The conversion of prochymosin to chymosin is favored by the low pH environment of the stomach.
Particular attention has been directed to studying the conversion of prochymosin to chymosin. Prochymosin was first reported by Hammersten in 1872 (Hammersten, O., Upsala Laekarefoeren. Foerh., 8, 63 (1872)). It is stable at neutral and alkaline pH and has been isolated by several methods.
Foltmann and coworkers have studied the conversion of natural prochymosin to chymosin (Pedersen, V. B. and Foltmann, B., Eur. J. Biochem. 55, 95 (1975)); (Foltmann, B. et al., Proc. Nat. Acad. Sci. (U.S.) 74, 2321 (1977)). (Pedersen, V. B., Christensen, K. A. and Foltmann, B., J. Biochem. 94, 573 (1979); Foltmann, B., C. R. Trav. Lab. Carlsberg, 35, 143 (1966)). They proposed that at pH2, an intermolecular proteolysis occurs which cleaves the prochymosin at the Phe-Leu[27-28] bond, to produce an active form, "pseudochymosin", which converts to chymosin at pH 4.0 or greater by intramolecular proteolysis. Others have postulated an intramolecular reaction for the generation of pseudochymosin (Al-Janaki et al., J. Biol. Chem., 247, 46 28 (1972)).
With the advent of recombinant DNA technology, it has become possible to produce prochymosin by the expression, in a transformed microorganism, of cDNA encoding the amino acid sequence of prochymosin under the control of a promoter and regulatory sequence. Copending, commonly assigned U.S. patent application Ser. No. 511,766, abandoned, filed July 7, 1983, describes a method of producing chymosin which involves expressing a DNA sequence encoding a fusion protein. The fusion protein comprises an amino-terminal fragment of a microbial protein normally associated with the promoter sequence (e.g., the .beta. subunit of tryptophan synthetase) fused to "activatable prochymosin". The activatable prochymosin comprises the amino acid sequence of "mature" chymosin together with a sufficient portion of its prosequence (at least about 15 amino acid residues) such that the product can undergo post translational autocatalytic cleavage to produce active chymosin. In addition to a portion of the prosequence, the prochymosin fusion product can contain part or all of the signal peptide of preprochymosin and/or a short linker sequence of amino acids, the latter being situated between the amino-terminal fragment of the microbial protein and the amino acid residues of prochymosin (or preprochymosin). The linker sequence in the expressed protein results from the translation of a synthetic DNA linker sequence which serves to fuse the DNA sequence encoding the microbial protein fragment to the DNA sequence encoding prochymosin (or preprochymosin) in the expression vector and to keep the reading frames of the sequences encoding the two segments in phase.
The expression product, i.e., the prochymosin fusion protein, forms an insoluble precipitate within the cells. After cell lysis, this precipitate is both insoluble in conventional solvents for proteins and incapable of undergoing activation by autocatalytic cleavage to produce mature chymosin. The inability of the expression product to self-activate is believed to be related to improper folding of the expressed protein, which in turn is related to the unnatural environment of the microbial host in which it is expressed. Accordingly, there is needed a method of recovering and activating microbially produced prochymosin.